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S C Cole

Publications and source records attributed to S C Cole.

15 recordsLinked to original sources
PubMed1995

Amino-acid substitutions in the cleavage site of acyl-coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum: effect on proenzyme cleavage and activity.

Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102-->Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage.

Acyltransferases↗
PubMed1994

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity.

Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser227-->Cys had no effect, Ser227-->Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser309-->Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser309-->Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation.

Acyltransferases↗
PubMed1993

The requirement for subunit interaction in the production of Penicillium chrysogenum acyl-coenzyme A:isopenicillin N acyltransferase in Escherichia coli.

Subunit interaction in the formation of active acyl-coenzyme A:isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the alpha (11 kDa) and beta (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly102/Cys103. Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing subunits of 11 and 29 kDa (similar to wild-type AT). Complete separation of the alpha and beta subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A:6-aminopenicillanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing Thr105 with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subunit in E. coli produced acyl-CoA:6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.

Acyl Coenzyme A↗
PubMed1993

On the production of alpha, beta-heterodimeric acyl-coenzyme A: isopenicillin N-acyltransferase of Penicillium chrysogenum. Studies using a recombinant source.

A high level E. coli expression system has been constructed for the Penicillium chrysogenum penDE gene, which encodes the acyl-coenzyme A: isopenicillin N-acyltransferase (AT) enzyme. Induction of overexpression of recombinant AT (recAT) by increasing the growth temperature of the host adversely affected solubility and activity of the AT enzyme. Addition of isopropylthio-beta-D-galactopyranoside (IPTG) at decreased growth temperatures (less than 32 degrees C) resulted in the overproduction of soluble, active recAT. When purified to homogeneity, recAT was an alpha, beta-heterodimer, comprised of 11 kDa (alpha) and 29 kDa (beta) subunits, derived from a 40 kDa precursor polypeptide by a posttranslational cleavage. The recAT enzyme contained both the acyl-coenzyme A: isopenicillin N-acyltransferase and the acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase activities. The processing event that generated the two subunits of recAT from the 40 kDa precursor polypeptide occurred between Gly102/Cys103. This expression system produced a large amount of soluble, active recAT that is identical to native AT, making it a suitable source of AT enzyme for further characterization.

Acyltransferases↗
PubMed1988

Carrot cells detoxify N-phosphonoacetyl-L-aspartate by esterification.

Unlike bacterial and mammalian cells, carrot cells are able to tolerate N-phosphonoacetyl-L-aspartate (PALA), a potential inhibitor of pyrimidine biosynthesis, by detoxifying the compound. Anion-exchange chromatography showed that detoxified PALA was less negatively charged than PALA, and allowed detoxified PALA to be isolated. Incubation of detoxified PALA with a low-specificity carboxylic-ester hydrolase fully restored the ability to inhibit aspartate transcarbamoylase, the target enzyme, indicating that the detoxification involves the formation of carboxylic ester. G.1.c. analysis of the alcohol products of enzymic hydrolysis, and of their ratio to PALA, showed that the detoxification produced a mixture of mono- and di-carboxylic esters and of methyl and ethyl esters. The detoxification mechanism showed considerable specificity towards PALA, since the analogous carboxy groups of succinate were not modified in the same way. Succinate was depleted much more slowly, no succinate esters could be detected, and the presence of a 10-fold excess of succinate did not inhibit the esterification rate of PALA. The possible significance of these results is discussed.

Aspartate Carbamoyltransferase↗
PubMed1988

Natural head position, posture, and prognathism: the Chapman Prize Essay, 1986.

Natural head position and natural head posture are terms which have been used synonymously to describe the spatial relationships of the head with reference to the true vertical, the cervical column, or both. This is confusing, and it would seem logical to ascribe one term to one reference and consider the phenomena separately. For the purposes of this prospective cephalometric survey, natural head position will describe the relationship of the head to the true vertical, while natural head posture will describe the relationship of the head to the cervical column. In order to investigate these two relationships of the head, five groups were formed, three representing horizontal discrepancies selected on ANB differences (class I, II and III dental base relationships) and two representing vertical discrepancies selected on Bjork's posterior angles ('high angle' and 'low angle' groups). Despite the marked differences in 'skeletal' pattern shown between the five groups, the values obtained for the saddle angles (N-S-Ar) showed no significant difference. However, the natural head positions and natural head postures were significantly different (P less than 0.01). Natural head position affected cranial base orientation and this alone produced class II or III effects. Natural head position was also associated with maxillary prognathism whilst natural head posture appeared more closely related to mandibular prognathism. These phenomena may help to explain an apparent class II or III tendency in cases where the saddle angle is normal.

Adolescent↗
PubMed1987

Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5'-phosphate.

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].

Amino Acid Sequence↗
PubMed1986

Comparison of aspartate transcarbamoylases from wheat germ and Escherichia coli: functionally identical histidines in nonhomologous local sequences.

Aspartate transcarbamoylase (ATCase) from wheat germ and the catalytic subunit of the enzyme from Escherichia coli are trimers of similar size. The former is a regulatory enzyme in its trimeric state, while the latter is a component of a complex regulatory dodecamer. In a comparison of the two enzymes, reaction with diethyl pyrocarbonate revealed a highly active, essential histidine residue in each case. The two histidines (i.e., one in each enzyme) behaved nearly identically with respect to the following functional properties: kinetics of acylation (ethoxyformylation) and concomitant inactivation; kinetics of deacylation by hydroxylamine and concomitant reactivation; hyperbolic dependence of the apparent first-order rate constant (kapp) on diethyl pyrocarbonate concentration; pH dependence of kapp; failure of active-center ligands to protect the residue against diethyl pyrocarbonate, producing instead near-identical increases in the inactivation rate. These similarities point to an essential, highly conserved histidine in each enzyme, in a functional microenvironment that has changed relatively little since the divergence of plants and bacteria. Ethoxyformylated peptides were isolated from tryptic digests of the two inactivated enzymes. Sequencing of the major labeled peptide in each case showed the wheat and E. coli histidines embedded in nonhomologous primary segments, suggesting that, contrary to expectation, these segments are not part of the conserved microenvironment. In the case of the E. coli enzyme, the essential residue was identified as His-134 in the known sequence, which has a potential catalytic role on crystallographic evidence [Krause, K. L., Volz, K. W., & Lipscomb, W. N. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1643-1647]. A second, much less reactive histidine was identified as His-64.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Inactivation of wheat-germ aspartate transcarbamoylase by the arginine-specific reagent phenylglyoxal.

Wheat-germ aspartate transcarbamoylase (EC 2.1.3.2) was inactivated by phenylglyoxal in a first-order process, provided that the inactivation time did not exceed 10 min. Apparent first-order rate constants were linearly dependent on phenylglyoxal concentration, indicating a bimolecular reaction between a single active-centre residue and phenylglyoxal, with second-order constant of 0.023 mM-1 X min-1. A plot of apparent first-order rate constant versus pH showed a steep rise above pH 9.5, indicating that the essential residue has a pKa value of 10.5 or higher, consistent with an arginine residue. Saturating concentrations of the following ligands provided a degree of protection (percentages in parentheses) against 1 mM-phenylglyoxal: N-phosphonoacetyl-L-aspartate, a bisubstrate analogue (94%); carbamoyl phosphate (75%); UMP, an end-product inhibitor (53%). Succinate (an analogue of L-aspartate) alone gave no protection, but in combination with carbamoyl phosphate raised the protection to 92%, in agreement with the known binding order of the two substrates. These results indicate that the essential arginine residue is close to the carbamoyl phosphate site, probably oriented towards the aspartate site. Attempts to desensitize the UMP-binding site by reaction with phenylglyoxal, while protecting the active centre, were unsuccessful. The essential active-centre arginine residue is compared with a similar residue in the Escherichia coli enzyme.

Aldehydes↗
PubMed1984

Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility.

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.

Aspartate Carbamoyltransferase↗
PubMed1983

Poly(ethylene glycol) quantitation by laser nephelometry.

When poly(ethylene glycol) 3350 is estimated by the method of Skoog [(1979) Vox Sang. 37, 345-349], fine particles form. The particles are not attributable to residual protein but to a poly(ethylene glycol)/barium/iodine complex that can be quantitated by means of a laser nephelometer. The method is sensitive to at least 10 mg% poly(ethylene glycol) 3350 (4 micrograms in the cuvette) in 2500 mg% protein, and nephelometer response is approximately linear between 30 and 200 mg% of the polymer. The coefficient of variance is about 8%. Triton X-100, Pluronic F-68, Varonic 1000MS, and poly(ethylene glycol) of higher and lower molecular weight react well. Alkylated celluloses, dextrans, glycerol, glycine, and sodium dodecyl sulfate do not react significantly. Barium can be replaced with Mg, Ca, Ni, Fe, and other divalent cations in the reaction, but other than for Hg, light-scattering is most intense with Ba. The reaction goes to completion in about 5 min and is most intense when the barium is added before the iodine.

Chemical Phenomena↗
PubMed1981

Rapid high-performance liquid chromatographic method for the measurement of verapamil and norverapamil in blood plasma or serum.

A simple high-performance liquid chromatographic method for the simultaneous measurement of plasma verapamil and norverapamil concentrations has been developed. The sample (100 microliters) is vortex-mixed for 30 sec with 4 M sodium hydroxide solution, pH 13 (50 microliters), internal standard solution (aqueous 5,6-benzoquinoline, 0.20 mg/l) (50 microliters) and methyl tert.-butyl ether (200 microliters). After centrifugation at 9950 x g for 2 min, a portion (100 microliters) of the resulting extract is analysed on a microparticulate (5 microns) silica column using a methanolic solution of potassium bromide (3.0 mM) and perchloric acid (0.37 mM) as the mobile phase, and the column effluent is monitored by fluorescence detection using an excitation wavelength of 203 nm. A specimen, together with a quality control sample, can be analysed, in duplicate, within 30 min. The limit of accurate measurement of the assay is 2 micrograms/l, and no potential sources of interference have been identified. The method has advantages of speed, small sample requirement and complete resolution of the three major metabolites of verapamil.

Chromatography, High Pressure Liquid↗